Lawrence Hayward - US grants

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is an age-dependent degenerative disorder of motor neurons characterized by progressive weakness and death within five years after the onset of symptoms. A subset of familial ALS is caused by mutations in Cu/Zn superoxide dismutase (SOD1) that render this antioxidant enzyme toxic by an unknown mechanism. Expression of mutant SOD1 in man or mice causes selective degeneration of motor neurons in the brain and spinal cord. The long-term objectives of this study are to understand how mutant SOD1 kills neurons and to identify novel pathways of cell death that may be relevant to ALS. Aim 1 of this project is to define important biophysical and biochemical characteristics that distinguish mutant from wild type SOD1. Recent studies in our lab indicate that mutant SOD1 enzymes all share an increased tendency to unfold or to lose metal ions when stressed by denaturing influences. Experiments will be performed to (1) define conditions that preferentially destabilize mutant but not normal SOD1, (2) identify factors that alter the dynamics of the SOD1 monomer-dimer equilibrium as measured by analytical ultracentrifugation, and (3) map regions of the protein that are susceptible to proteolytic cleavage or partial unfolding. In Aim 2, the affinities and thermodynamics of metal binding to SOD1 as a function of pH or denaturant will be measured using a novel calorimetric approach. Conditions that alter Cu ion reactivity in mutant SOD1 will also be defined. Aim 3 will employ cell culture models to test hypotheses of mutant SOD1 toxicity that are consistent with the physicochemical findings from Aims 1 and 2. Initial investigations will test whether early lysosomal dysfunction occurs in response to specific stresses in mutant SOD1 -bearing neuroblastoma cells or in organotypic spinal cord slice cultures. Future results from Aims 1 and 2 may favor alternative hypotheses of mutant SOD1 toxicity to test in these cellular models and may suggest new therapeutic approaches to evaluate in ALS mice.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15-NS-104: Early-stage Therapy Development. Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is a devastating neurodegenerative condition that kills nerve cells in the brain and spinal cord that control the muscles, leading to progressive weakness and death within 3-5 years. Currently, no treatment can slow the progression of the disease, thus raising the urgency for developing new animal models of ALS that can be used to identify novel therapeutic strategies. Recently mutations in a new gene, FUS/TLS, have been identified to cause ~5% of familial ALS cases. The mechanism(s) by which these mutations cause the disease is not clear, but they may overlap with defects in other known ALS genes such as TDP-43, senataxin, and dynactin. These genes may function in the processing, delivery, or regulation of RNA molecules, and thus, defects in these functions may underlie motor neuron vulnerability in ALS. The direct causal link of mutant FUS/TLS to ALS provides an opportunity to develop novel in vivo ALS models that will accelerate the identification of new treatment target(s) for ALS. To generate new and informative ALS models rapidly based on FUS/TLS mutations, we propose to use three parallel but complementary approaches to express normal or mutant FUS/TLS in transgenic mice. In the first approach, we will produce mice in which expression of FUS/TLS (wild type and two ALS mutants) is driven by native genomic regulatory elements. A novel aspect of our design will be the incorporation of conditional knockout capabilities that will allow us to selectively turn off the mutant gene expression to determine in which cell types the mutant proteins exert their most potent effects. In the second approach, we will generate mice in which expression of FUS/TLS transgenes is activated only after crossing with mice that express Cre recombinase in specific tissues. This strategy will allow the selection and breeding of founder animals even if the mutant gene is highly toxic. Furthermore, we can determine whether transgene expression causes motor neuron toxicity by cell autonomous or non-cell autonomous mechanisms. In the third approach, we will express wild type and mutant FUS/TLS in transgenic mice under temporal control using a tetracycline-inducible strategy. In the case of developmental or early postnatal toxicity of transgene expression, this approach will allow us to initiate or silence expression at different ages, thus enabling us to investigate the role of aging in this disease and the reversibility of this disease when the mutant gene is silenced. These studies will guide the development of therapies in the future. Together, these complementary approaches are likely to produce one or more animal models of ALS based on FUS/TLS mutations, making them available for us and the research community in general to study the mechanism of the disease and to develop new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is a devastating neurodegenerative condition in which the nerve cells in the brain and spinal cord that control the muscles die prematurely. Currently no truly effective treatments exist to slow the relentless course of the disease. This project will address the urgent need to develop informative mouse models of ALS that will i) accelerate the identification of novel treatment target(s) and ii) enable the testing of new therapeutic strategies to combat ALS.

Abstract Dominant mutations in the gene encoding the nucleic acid binding protein FUS cause ~5% of familial amyotrophic lateral sclerosis (ALS). Our long-term objectives are to discern the mechanism(s) by which FUS mutants injure aging motor neurons and to develop novel therapeutic approaches to increase the defenses against these insults. Our laboratory has identified a novel and robust nuclear phenotype caused by ALS- linked FUS mutants: impaired stress-responsive processing of sub-nuclear assemblies known as promyelocytic leukemia (PML) nuclear bodies. PML nuclear bodies are induced by a variety of cellular stresses and regulate nuclear protein homeostasis, transcription, DNA-damage pathways, and cellular senescence, yet their potential role in ALS has not been explored. We observed that PML bodies were abnormally enlarged both in cell lines and in primary ALS human fibroblasts expressing mutant FUS. Furthermore, proteasome activities were decreased, and exposure to mild oxidative stress or proteasome inhibition in FUS mutant but not control cells stalled the turnover of expanded PML bodies. We hypothesize that the observed abnormality of PML nuclear bodies may report upon altered nuclear homeostasis resulting from FUS mutant expression. In Aim 1 of this project, we will develop an imaging-based phenotypic screening assay to identify small molecule compounds that modulate the observed PML nuclear body enlargement in cells expressing FUS mutants. We will test compound libraries that include FDA-approved drugs and diverse CNS-active agents predicted to cross the blood-brain barrier. We will prioritize initial hits using dose-response studies and will determine whether proteins known to be targeted to the proteasome following stress are more effectively eliminated upon treatment with hit compounds. We have established transgenic mice harboring ALS-linked FUS variants and have observed a phenotype of age-dependent loss of the connection between motor nerves and muscle in mice that express the R495X mutant. In Aim 2 of this project, we will validate hit compounds and analyze pathways related to PML nuclear body function in CNS cells and tissues from our mutant FUS transgenic mice. We will test whether a subset of the prioritized hit compounds from Aim 1 ameliorate defects of nuclear protein homeostasis or proteasome activity in transgenic primary cortical neurons, glial cells, or motor neurons. In further experiments, which do not depend upon the success of obtaining modulator compounds in Aim 1, we will define more precisely which pathways related to PML nuclear body function are most relevant to FUS- mediated ALS. We will purify proteasomes from the CNS of our FUS mice at pre-symptomatic and symptomatic ages and will quantify subunit expression, assembly, and activities in collaboration with Dr. Fred Goldberg's group. We will also use a high-resolution tissue monolayer preparation to identify nuclear insults related to altered PML body function in aging neurons from our FUS transgenic mice.